The signaling phospholipid PIP2 has been recently appreciated for its critical role in regulating the activity of many ion channel and transporter proteins. The molecular details of how PTP2 controls the activity of such a diverse number of transmembrane proteins are unclear. Recent advances in the elucidation of the three-dimensional structure of a number of ion channel proteins makes it possible to study this problem at an atomic level of detail. Here we propose to identify the two end points of K channel gating by PIP2: the channel interaction sites with PTP2 and the channel gate that they control. First we will identify the channel gate that PIP2 controls in inwardly rectifying K (Kir) channels by using electrophysiology and mutagenesis coupled with a Substituted Cysteine Accessibility Method (SCAM) to probe state dependent modification during channel gating by PIP2. Secondly we will study the allosteric effects of Na gating in Kir3 and other Kir channels and the atomic details of Kir channel-PIP2 interactions. These studies will enable us to understand such functional consequences of these interactions, as the apparent channel affinity for PIP2 and the phosphoinositide stereospecific interactions with distinct channel sites. In close collaboration with computational biologists in our department, we have developed a combined electrophysiological and theoretical approach to analyze PIP2-sensitive channels: we first define their PIP2 interacting regions by computing molecular interaction fields of channels with phosphate probes;using a complementary computational approach we run Brownian Dynamics (BD) simulations using phosphoinositide probes to define the PIP2 interacting residues. We compare these two computational approaches and their agreement justifies pursuit of analysis at the next level, namely the refinement of the BD structures by Molecular Dynamics simulations to generate reliable models that can be tested experimentally. The validity of this combined experimental/theoretical approach has been developed and reinforced in Kir channels, where we have generated laborious experimental evidence of amino acid residues involved in interactions with PIP2. In the present competitive continuation of this work we propose to extend these studies to the family of voltage-gated K (Kv) channels, particularly because of the availability of a new Kv1.2 crystal structure. We provide preliminary data to show that different Kv subfamily members are PIP2-sensitive and propose a similar characterization of PlP2 sensitivity as with the Kir channels. Renewal of this grant award will allow us to gain mechanistic insights into how different channel superfamilies are dependent on PIP2.